There are two important techniques for separation of biomolecules. Chromatography is generally used for a preparative purification of biological molecules, whereas electrophoresis is the most powerful technique for analysis of the molecules in crude samples and at various stages of a purification procedure.
Each of these two techniques uses separation media of unique properties. Nevertheless, media for both or either chromatography and electrophoresis can be prepared from the same starting material. Until now, only two acrylic monomers have been successfully used to prepare gels for electrophoresis and chromatography. These two monomers are acrylamide and N-acryloyl-tris(hydroxymethyl)-aminomethane (NAT). One of the inventors of the present invention is one of the persons who have introduced the NAT-monomer to prepare poly-NAT gels for electrophoresis (Kozulic, M., Kozulic, B., and Mosbach, K. (1987), R. Anal. Biochem. 21 26 23 2, 2, 506-512 (reference 1); Kozulic, B., Mosbach, K., and Pietrzak., M. (1988), Anal. Biochem. 21 27 20 2, 2, 478-484 (reference 2); and International Patent Application No. PCTEP88/00515) (Reference 3).
The poly-NAT gels possess several advantages over the polyacrylamide gels. In addition to their pronounced hydrophilicity, their most important advantage is the higher porosity of these gels. Since a gel even more hydrophilic and porous than a poly.NAT gel would be beneficial in many applications, a search was made for a monomer which could be polymerized to produce such a gel.
A NAT solution has a molar concentration lower than a polyacrylamide solution of the same weight percentage, because the molecular weight of NAT is about 2.5 fold higher than the molecular weight of acrylamide. The poly-NAT gels have been found to be approximately 3 times more porous than the corresponding polyacrylamide gels, which is in good agreement with the 2.5 fold lower molarity. Thus, one can assume that the lower molar concentration of NAT solutions results, after polymerization, in fewer polymer chains per unit volume, leading to gels of increased porosity. If this assumption is correct, then even more porous gels will be formed from monomers of even higher molecular weight. However, the lack of mechanical strength may be a drawback of gels produced from monomers of very high molecular weight. The optimal properties are expected to be inherent to the monomers of medium size, due to a balanced ratio between the size of the polymer backbone and the size of the side chains present in every repeating unit.
In addition to the size of a monomer, other factors can influence the porosity of a gel. If interactions exist between monomer molecules, or between a monomer and a growing polymer, or between the two growing polymer chains, then as a result of these interactions the polymer chains will not be randomly distributed. They are likely to form some kind of bundles and thus create large pores. It is generally accepted that the high porosity of agarose gels comes from an association of polymer chains during the gelation process. The resulting bundles of polymer chains are presumably held together by hydrogen bonds. Other types of stabilizing forces, such as hydrophobic interaction or ionic bonds, are not compatible with the media that are to be used for electrophoresis or chromatography.
From the above considerations, it appeared that monomers composed of sugar alcohols might form gels with desirable properties. They are hydrophilic, they are of medium size and they have 4-5 hydroxyl groups which could form hydrogen bonds. As shown in the present invention, many such compounds can be conveniently and controllably synthesized. In addition, such synthesized compounds easily polymerize and form gels useful for electrophoresis and chromatography. Two monomers of the general type described herein, N-acryloyl-1-amino-1-deoxy-D-glucitol and N-methacryloyl-1-amino-1-deoxy-D-glucitol, have been synthesized previously (Whistler, R.L., Panzer, H.P., and Roberts, H.J. (1961), J. Org. Chem. Vol. 26 p. 1583-1588), but they were not used to prepare crosslinked gels for electrophoresis or chromatography.